The HIV-1 pathogenic factor Nef and its interactions with host proteins are important for viral pathogenesis, but their role in HIV-1 latency is not known. Nef-associated factor 1 (Naf1), also known as A20-binding inhibitor of NF-kB (ABIN1), is a host protein that inhibits NF-kB activation. NF-kB is a well-known regulator HIV-1 gene expression and viral latency. Intriguingly, our preliminary studies suggest that Naf1 and Nef interplay regulates HIV-1 gene transcription and viral latency. We found that (1) Naf1 suppresses NF-kB- dependent HIV-1 gene expression and Nef counteracts the effects; and (2) Naf1 maintains HIV-1 latency by suppressing viral gene transcription, while Nef antagonizes the effects. Therefore, we propose to delineate the mechanisms of Nef-Naf1 interactions in regulating HIV-1 latency and to seek a new approach to overcome viral latency. Our central hypotheses are (1) Naf1 maintains HIV-1 latency by suppressing NF- kB-dependent viral gene transcription; (2) Nef antagonizes the inhibitory effects of Naf1 by inducing Naf1 phosphorylation, which activates PI3K/Akt signaling and leads to HIV-1 reactivation from latency. We propose two specific aims to test these novel hypotheses. Aim 1. To investigate the mechanisms by which the Naf1-Nef interaction regulates HIV-1 gene expression. Aim 2. To determine the function of Naf1 and Nef interactions in modulating HIV-1 latency in primary CD4+ T cells. Accomplishing the proposed studies through our collaborative efforts will provide novel insights into the viral and cellular mechanisms of modulating HIV-1 latency. Our expected results will provide a basis to target Nef-Naf1 interactions and to enhance anti-retroviral therapy toward a functional cure for HIV-1/AIDS.